In general, the invention relates to methods for treating a neurological disease.
Neurological diseases, for example, Alzheimer""s disease, are often difficult to diagnose and occur in the population in a manner which is difficult to predict. A method that would allow one to identify subjects having a neurological disease, or being at risk for developing a neurological disease, would allow for the more timely administration of an appropriate therapy.
The GPIIIa gene encodes a 788 amino acid polypeptide with a 26-residue signal peptide, a 29-residue transmembrane domain near the carboxy terminus, and four cysteine-rich domains of 33-38 residues each (Zimrin et al., J Clin. Invest. 81:1470-1475 (1988)). Two different antigenic forms of GPIIIa, alloantigens PlA1 and PlA2 (for Platelet Antigen 1 and 2), have been described and can be distinguished using a monoclonal antibody (Weiss et al., Tissue Antigens 46:374-381 (1995)). The most predominant form of GPIIIa, PlA1, is carried by 98% of the Caucasian population. The rarer form of GPIIIa, PlA2, has sustained a point mutation at base 192 that causes a nucleotide change from a T to a C and thus a leucine to proline (CTG greater than CCG) amino acid substitution at residue position 33 (Newman et al., J. Clin. Invest. 83:1778-1781 (1989)).
The GPIIb polypeptide is the larger component of the GPIIIa/GPIIb complex and comprises two disulfide-linked subunits of 137 amino acids and 871 amino acids each. The larger GPIIb polypeptide has a 26 amino acid signal sequence, a potential transmembrane domain, and four stretches of 12 amino acids each that are homologous to the calcium binding sites of calmodulin and troponin C (Poncz et al., J Biol. Chem. 262(18):8476-8482 (1987)). Mutational analysis of these domains has indicated that these calcium-binding domains are required for the correct folding and transport of the GPIIb polypeptide to the cell surface (Basani et al., Blood 88:167-173 (1996)). Two antigenic forms of GPIIb, Baka and Bakb, have been described and can be distinguished using specific antisera. The less common form of GPIIb (i.e., Bakb) was determined to have a T to G point mutation that results in an isoleucine to serine substitution at amino acid position 843 (Lyman et al., Blood 75:2343-2348 (1990)).
The present invention provides methods for identifying or treating a subject at risk for, or diagnosed with, a neurological disease.
In the first aspect, the invention provides a method for identifying a subject at risk for a neurological disease by: identifying the subject; determining the genotype or phenotype of the GPIIIa or GPIIb locus of the subject; and determining the presence of a variant GPIIIa or a variant GPIIb allele or isoform, where the presence of the variant GPIIIa allele or isoform or the variant GPIIb allele or isoform is indicative of the subject having an increased risk of the neurological disease. Preferably, the neurological disease is Alzheimer""s Disease (AD).
In the second aspect, the invention provides a method for diagnosing a subject with a neurological disease by: identifying the subject; determining the genotype or phenotype of the GPIIIa or GPIIb locus of the subject; and determining the presence of a variant GPIIIa or a variant GPIIb allele or isoform, where the presence of the variant GPIIIa allele or isoform or the variant GPIIb allele or isoform is indicative of the subject having a likelihood of the neurological disease.
In the third aspect, the invention provides a method for characterizing the genotype of at least one subject involved in a clinical trial of a therapy for the treatment of a neurological disease by: identifying the subject; determining the genotype or phenotype of the GPIIIa or GPIIb locus of the subject before, during, or after the clinical trial; and determining the presence of a variant GPIIIa or a variant GPIIb allele or isoform, where the presence of the variant GPIIIa allele or isoform or the variant GPIIb allele or isoform places the subject into a subgroup for the clinical trial. Preferably, the genotype or phenotype is indexed against the efficacy or side-effects of the therapy.
In the fourth aspect, the invention provides a method for treating a subject with a neurological disease by: identifying the subject; determining the genotype or phenotype of the GPIIIa or GPIIb locus of the subject; determining the presence of a variant GPIIIa or a variant GPIIb allele or isoform; and determining the preferred therapy for the treatment of the neurological disease.
In the fifth aspect, the invention provides a method for treating a subject at risk for a neurological disease by: identifying the subject; determining the genotype or phenotype of the GPIIIa or GPIIb locus of the subject; determining the presence of a variant GPIIIa or a variant GPIIb allele or isoform; determining the GPIIIa or GPIIb allele status of the subject, where the allele status is predictive of patient outcome or drug efficacy.
In a preferred embodiment of the above aspects, the method includes determining the presence of both the variant GPIIIa allele or isoform and the variant GPIIb allele or isoform.
In other preferred embodiments of the above aspects, the neurological disease may be Alzheimer""s disease (AD), a non-AD neurological disease, or a neurological disease selected from the group consisting of Alzheimer""s disease, neurofibromatosis, Huntington""s disease, depression, amyotrophic lateral sclerosis, multiple sclerosis, stroke, Parkinson""s disease, and multi-infarct dementia.
In other preferred embodiments of the above aspects, the determining may be performed using a nucleic acid that specifically binds a nucleic acid encoded by the variant GPIIIa allele or the variant GPIIb allele. In other preferred embodiments of the above aspects, the determining may be performed using an antibody that specifically binds a polypeptide encoded by the variant GPIIIa allele or the variant GPIIb allele, but does not bind a polypeptide encoded by a wild-type GPIIIa allele or a wild-type GPIIb allele.
In other preferred embodiments of the above aspects, the variant GPIIIa allele may have a point mutation at nucleotide base 192 of SEQ ID NO: 2 or encode a polypeptide with a proline at amino acid position 33 of SEQ ID NO: 4. In other preferred embodiments of the above aspects, the variant GPIIb allele may have a point mutation at nucleotide base 2622 of SEQ ID NO: 6 or encode a polypeptide with a serine at amino acid position 843 of SEQ ID NO: 8.
The presence of a variant allele may be determined by genotyping nucleic acids from the subject or by assaying for the presence of a protein having alterations encoded by the variant nucleic acid.
By xe2x80x9cneurological diseasexe2x80x9d is meant a disease, which involves the neuronal cells of the nervous system. Specifically included are: prion diseases (e.g, Creutzfeldt-Jakob disease); pathologies of the developing brain (e.g., congenital defects in amino acid metabolism, such as argininosuccinicaciduria, cystathioninuria, histidinemia, homocystinuria, hyperammonemia, phenylketonuria, tyrosinemia, and fragile X syndrome); pathologies of the mature brain (e.g., neurofibromatosis, Huntington""s disease, depression, amyotrophic lateral sclerosis, multiple sclerosis); conditions that strike in adulthood (e.g. Alzheimer""s disease, Creutzfeldt-Jakob disease, Lewy body disease, Parkinson""s disease, Pick""s disease); and other pathologies of the brain (e.g., brain mishaps, brain injury, coma, infections by various agents, dietary deficiencies, stroke, multiple infarct dementia, and cardiovascular accidents).
By xe2x80x9ccognitive enhancersxe2x80x9d is meant drugs which enhance a) memory performance, whether it is verbal memory, spatial memory, or factual memory and b) learning capacity.
By xe2x80x9ccholinomimetic therapyxe2x80x9d is meant any drug that mimics the function of acetylcholine or enhances the activity of acetylcholine synthesizing cells. These drugs include, but are not limited to, inhibitors of acetylcholine degradation (acetylcholine esterase inhibitors such as tacrine), drugs that mimic acetylcholine structure and function, drugs that block acetylcholine uptake by neurons, and drugs that interact with pre-synaptic receptors to induce acetylcholine release from cholinergic neurons.
By xe2x80x9cnon-cholinomimetic vasopressinergic therapyxe2x80x9d is meant a therapy that utilizes a vasopressinergic modulator such as, for example, S12024 (provided by Servier, Les Laboratoires Servier, 22 rue Gamier, 92200 Neuilly sur Seine, France).
By xe2x80x9calready diagnosedxe2x80x9d is meant already diagnosed as having the neurological disease, having a genetic predisposition to the disease, or both.
By xe2x80x9cpatient profilexe2x80x9d is meant data pertaining to the patient for whom the pharmacogenetic analysis is being performed. Data may include information on the patient""s diagnosis, age, sex, and genotype. The patient""s profile may also include materials from the patient such as blood or purified RNA or DNA.
By xe2x80x9cprognosis protocolxe2x80x9d is meant a therapy plan provided to the clinician or patient using the pharmacogenetic method. The prognosis protocol includes an indication of whether or not the patient is likely to respond positively to a cholinomimetic therapeutic. In preferred embodiments, the protocol also includes an indication of the drug dose to which the patient is most likely to respond. The xe2x80x9cpharmacogenetic methodxe2x80x9d is a method whereby genetic and diagnostic data, including the patient""s neurological diagnosis and the patient""s GPIIIa and/or GPIIb genotype are processed to provide therapeutic options and prognoses.
By xe2x80x9cnon-AD neurological diseasexe2x80x9d is meant a disease other than Alzheimer""s disease, which involves the neuronal cells of the nervous system. Specifically included are: prion diseases (e.g, Creutzfeldt-Jakob disease); pathologies of the developing brain (e.g., congenital defects in amino acid metabolism, such as argininosuccinicaciduria, cystathioninuria, histidinemia, homocystinuria, hyperammonemia, phenylketonuria, tyrosinemia, and fragile X syndrome); pathologies of the mature brain (e.g., neurofibromatosis, Huntington""s disease, depression, amyotrophic lateral sclerosis, multiple sclerosis); conditions that strike in adulthood (e.g. Creutzfeldt-Jakob disease, Lewy body disease, Parkinson""s disease, Pick""s disease); and other pathologies of the brain (e.g., brain mishaps, brain injury, coma, infections by various agents, dietary deficiencies, stroke, multi-infarct dementia, and cardiovascular accidents).
By xe2x80x9cAlzheimer""s Diseasexe2x80x9d is meant a pathology characterized by an early and extensive loss of entorhinal cortex neurons. Alzheimer""s disease subjects may be identified by progressive and degenerative effects on the brain which are not attributable to other causes. A diagnosis of Alzheimer""s disease is made using clinical-neuropathological correlations known in the art (see e.g., Arch. Neurology 51(9):888-896 (1994)). Post-mortem, the disease may be diagnosed by the presence of amyloid plaques and fibrils.
As used herein, by xe2x80x9ctherapy for the treatment of a neurological diseasexe2x80x9d is meant any therapy suitable for treating a neurological disease. A suitable therapy can be a pharmacological agent or drug that may enhance or slow the loss of cognitive function, motor function, or neuronal activity of the central nervous system, peripheral nervous system, or inhibit the further deterioration of any of these faculties. In addition, the term therapy may also include the close monitoring of an asymptomatic patient for the appearance of any symptoms of a neurological disease.
By xe2x80x9cdetermining the presence of a variant GPIIIa and/or variant GPIIb allelexe2x80x9d is meant subjecting a nucleic acid sample to any of a variety of detection techniques known in the art for elucidating a point mutation in a nucleic acid (e.g., polymerase chain reaction (PCR), reverse transcriptase-PCR (RT-PCR), ligase-mediated chain reaction step, chip hybridization methods, or restriction enzyme-mediated digestion). For example, in the presence of appropriately designed primers, a nucleic acid fragment can be amplified using PCR and analyzed by restriction enzyme digestion that can reveal the presence of a variant allelic sequence. In addition, DNA sequencing may be employed using techniques known in the art. These nucleic acid techniques allow for a genotype determination of the GPIIIa or GPIIb locus. Alternatively, phenotyping of the locus may be performed (and a genotype thus inferred) by using standard techniques for detecting the presence of a polypeptide having a particular amino acid change (e.g., antibodies, isoelectric focusing, and 2-D PAGE). For example, the presence of a variant GPIIIa polypeptide (e.g., PlA2; LEU33PRO) can be distinguished from a wild-type GPIIIa polypeptide (i.e., PlA1) using epitope specific antibodies available in the art (Weiss et al., Tissue Antigens 46:374-381 (1995)). Antibodies for detecting different polymorphisms of the GPIIb polypeptide have also been described (Lyman et al., Blood 75:2343-2348 (1990)).
By xe2x80x9cvariant GPIIIa allelexe2x80x9d is meant any sequence mutation of the glycoprotein integrin beta-3 subunit (GPIIIa) gene, that differs from the predominant wild-type allelic sequence (e.g., variant GPIIIa allele (LEU33PRO)) and which is associated with neurological disease. By xe2x80x9cassociatedxe2x80x9d is meant associated with an altered risk of disease incidence, drug efficacy, or disease prognosis. Variant GPIIIa alleles not specifically described to be associated with neurological disease herein can be tested for association using the techniques provided herein and those known in the art. Specifically excluded are GPIIIa variants that have an A greater than C mutation at nucleotide base 1159, and A greater than G mutation at nucleotide base 1549, or a G greater than C mutation at nucleotide base 1161.
By xe2x80x9cvariant GPIIb allelexe2x80x9d is meant any sequence mutation of the glycoprotein integrin alpha-2 subunit (GPIIb) gene that differs from the predominant wild-type allelic sequence (e.g., variant GPIIb allele (ILE843SER)) and which is associated with neurological disease. By xe2x80x9cassociatedxe2x80x9d is meant associated with an altered risk of disease incidence, drug efficacy, or disease prognosis. Variant GPIIb alleles not specifically described to be associated with neurological disease herein can be tested for association using the techniques provided herein and those known in the art. Specifically excluded are GPIIIa variants that have an A greater than C mutation at nucleotide base 1159, and A greater than G mutation at nucleotide base 1549, or a G greater than C mutation at nucleotide base 1161.
By xe2x80x9crisk factor associated with a diseasexe2x80x9d is meant any risk factor for a disease known in the art. Examples of risk factors commonly associated with diseases include age, gender, diet, exercise, weight, the presence of another disease, and the occurrence of a specific genotype. Risk factors associated with a neurological disease in particular may include advanced age, lower intelligence, smaller head size, history of head trauma, mutations on chromosomes 1, 14, and 21, or the presence of a variant GPIIIa and/or variant GPIIb allele (see e.g., Cummings et al., Neurology (1 Supp.1):S2-S17, 1998).
By xe2x80x9csubject at risk for a neurological diseasexe2x80x9d is meant a subject identified or diagnosed as having a neurological disease or having a genetic predisposition or risk for acquiring a neurological disease using the methods of the invention and techniques available to those skilled in the art.
By xe2x80x9cwild-typexe2x80x9d is meant any allele, or polypeptide encoded by such an allele, that is present in that part of the population considered free of disease.
By xe2x80x9cPCR, RT-PCR, or ligase chain reaction amplificationxe2x80x9d is meant subjecting a DNA sample to a Polymerase Chain Reaction step or ligase-mediated chain reaction step, or RNA to a RT-PCR step, such that, in the presence of appropriately designed primers, a nucleic acid fragment is synthesized or fails to be synthesized, thereby revealing the allele status of a patient. The nucleic acid may be further analyzed by DNA sequencing using techniques known in the art.
The present invention provides a number of advantages. For example, the methods described herein allow for a determination of a subject""s GPIIIa and/or GPIIb genotype for the timely administration of a prophylactic therapy for the treatment of a neurological disease.
Other features and advantages of the invention will be apparent from the following detailed description and from the claims.
The drawings will first be described.